This invention relates to a process for storing by freezing and subsequently recovering meristematic plant tissue as from the shoot apex having chlorophyll synthesis ability and morphogenic potential.
Unorganized callus tissues derived from plants have been propogated and differentiated into plants under the influence of growth hormones, however, such tissues exhibit genetic instability, chromosomal abnormalities, and often lose the ability to initiate organs after numerous subculturing steps. Since a plant obtained from such a culture is not necessarily identical to the parent, such a technique cannot be used as a propogation or storage method where identical plants are needed or desired. In addition, development, if it can take place in callus and cell cultures derived from callus or other plant tissue, also involved adventitious organogensis of either shoots or asexual embryos which can foster a high incidence of genetically aberrant plants.
Procedures for propogating and thus storaging plants involving serial transfer or subculturing of meristematic tissue as in adventitious or auxiliary shoot multiplication wherein the cultures are grown on a suitable growth medium are disadvantageous since eventual chromosomal changes occur so that the desirable characteristics of the parent plant cannot be obtained. Also, subcultures are lost through equipment failures. In addition, the tissue explants are subject to accidental contamination by microorganisms after extended subculturing.
Plants also are presently propogated from meristematic tissue by excising it from a donor plant and placing it in a growth medium. In some cases, as with pineapple or strawberry meristematic tissue, the explant is placed in a medium or under low above freezing temperatures (minimal growth conditions) which promotes only slow growth. The process requires that liquid medium be added to this tissue every few months to prevent dessication, thus exposing the cultures to microorganism contamination. In addition, using present processes, meristematic tissue cannot be stored indefinitely without its growing to a degree where the growth must be continued to maturity to sustain the plant. Therefore, the effective storage time for such tissue is greatly and undesirably limited.
Prior to the present invention, plant material has been frozen for storage and subsequently thawed while maintaining cell viability. Plant material processed in this manner includes cell culture cells, asexual embryos obtained from cell cultures and callus. While cell cultures are amenable to breeding by the fusion of haploid or diploid cells from different plants, in most species great difficulty is encountered in obtaining differentiation to a whole plant. Also, cell cultures can foster the same high incidence of genetically aberrant plants as do callus cultures. In addition, asexual embryos are not formed in a wide variety of plant species. Finally, the induction, maintenance and freezing of cell or embryo cultures at low freezing rates and recovery, thereof, requires a great deal of specialized skill and equipment.
Accordingly, present processes for storing plant cultures are deficient in that there are no reliable methods which permit initiation of growth of daughter plants in a wide variety of plant species for long periods after initial derivation of tissue from the parent plant and for assuring exact genetic duplication of parent plants which are essentially pathogen-free.